PROJECT SUMMARY/ABSTRACT Unbalanced bone remodeling often causes bone loss and leads to osteoporosis. Wnt/?-catenin signaling is essential for commitment and differentiation of bone forming osteoblasts from mesenchymal progenitors. Changes in mechanical loading can also influence bone formation. Osteocytes, former osteoblasts buried in bone matrix, are thought to be the cells that perceive mechanical signals and orchestrate bone remodeling. However, the molecular mechanisms by which osteocytes perceive and transduce mechanical signals are not fully understood. YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif), two related transcriptional co-factors that shuttle between the cytoplasm and the nucleus, have emerged as potentially important transcriptional regulators in mechanotransduction. Their activity is dependent on their subcellular localization, which is tightly regulated by different extracellular cues. Cytoplasmic YAP and TAZ promote proteosomal degradation of ?-catenin and thereby inhibit Wnt signaling. Mechanical signals emanating from rigid extracellular matrix or from fluid flow promote YAP and TAZ translocation into nucleus where they stimulate transcription. Our preliminary studies show that deletion of YAP and TAZ from osteoblast progenitors increased osteoblast formation in mice, and this was associated with increased Wnt signaling. In contrast, deletion of YAP and TAZ from differentiated osteoblasts and osteocytes decreased osteoblast number and bone formation. These results suggest that YAP and TAZ perform different functions in mesenchymal progenitors versus mature osteoblasts and osteocytes. Based on this, we hypothesize that the less rigid environment of osteoblast progenitors retains YAP and TAZ in the cytoplasm, promotes ?-catenin degradation, and thereby inhibits osteoblast differentiation. In contrast, the more rigid environment of osteoblasts and osteocytes, together with other mechanical inputs (such as fluid shear), favors nuclear retention of YAP and TAZ and thereby increases osteoblast number. To test this hypothesis, we will determine whether YAP and TAZ in osteoblast progenitors inhibit their differentiation by promoting ?-catenin degradation using genetically modified mice in which YAP will be restricted to the cytoplasm or the nucleus (Aim 1). In addition, we will examine whether YAP and TAZ expression in osteocytes mediates the effects of changes in mechanical loading on osteoblast number using tail suspension and cyclic compression models (Aim 2). Further, we will perform in vitro studies to identify YAP and TAZ target genes in osteoblasts and osteocytes that are responsible for their effects on osteoblast number using genome-wide mRNA expression analysis (RNA-seq) in osteoblastic and osteocytic cells before and after suppression of the endogenous YAP and TAZ genes (Aim 3). Successful completion of these studies will shed light on novel mechanisms that control bone formation and will advance knowledge of how the skeleton responds to changes in mechanical load.